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dc.contributor.authorDeal, Parker
dc.contributor.authorAl Abdullatif, Sarah
dc.contributor.authorMiller, Evan
dc.date.accessioned2019-06-26T11:38:14Z
dc.date.available2019-06-26T11:38:14Z
dc.date.issued2019-01-13
dc.identifier.urihttp://hdl.handle.net/10754/655717
dc.description.abstractSynthesis of Red-Shifted Flourescent Voltage Sensors Sarah Al Abdullatif; Parker Deal; Prof. Evan Miller UC Berkeley, Department of Chemistry Introduction Fluorescence imaging can be used to monitor electrical activity in neurons by using voltage sensitive dyes (VSDs). VSDs allow the measurement of transmembrane potentials through a photo-induced electron transfer (PeT) from an electron donor through a molecular wire to a uorophore. At resting or hyperpolarized potentials, the transmembrane electric eld promotes PeT, quenching the excited-state uorophore. Depolarization reverses the electric eld, decreasing the rate of PeT, and therefore leading to an increase of uorescence. Recently we reported the development of a new class of VFs based on isomerically pure tetramethylrhodamines. These new Rhodamine Voltage Reporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing to measure changes in membrane potential with high sensitivity (up to 47% F/F per 100 mV). In addition, RhoVRs possess excitation and emission proles in the green to orange region of the visible spectrum, allowing for use alongside commonly used green uorescent tools such as GFP. Modications to RhoVR Piperazine Functionalized Voltage Reporters Functionalization of RhoVR dyes with L-cysteic acid functionalized piperazine maintains a tertiary amide at the 2’ position, includes a sulfonate to aid with solubility and orientation of the dye in the membrane , and provides a nucleophilic handle for attachment of targeting ligands. In addition to modications at the 2' position, a new RhoVR, RhoVR(Me), was synthesized with a less electron rich wire that is complementary dye to RhoVR(OMe) as it is much brighter, however less sensitive, than the parent VF. Genetic Targeting Using HaloTag RhoVRs have demonstrated the ability to record neuronal activity with good signal to noise ratio, however the dyes indiscriminately label all membranes, leading to high levels of background uorescence. We have chosen to utilize HaloTag® technology as a means to address the limitations of RhoVRs through genetic targeting. HaloTag® is a dehalogenase-based labeling system which forms a covalent linkage between a protein tag and a chemical ligand (chloroalkane). By expressing Halotag extracellularly, then adding RhoVR linked to the HaloTag® ligand via a exible linker (PEG), we hope to gain the selectivity of genetically encoded voltage indicators (GEVIs) while maintaining the favorable photophysical properties of the small-molecule VF. PEG linkers of varying length were also investigated to determine if linker length aected the voltage sensitivity of the dyes. Genetic Targeting and Voltage Sensitivity The voltage sensitivites of the RhoVR-PEGn-Halo compounds were determined using patch-clamp electrophysiology. All derivatives were capable of measuring membrane potential, but also showed attenuated voltage sensitivities from the parent sarcosine-functionalized compounds. In addition the RhoVR-PEGn-HaloTag® compounds were roughly one third as bright as the parent dyes. Future Work Future work with RhoVR-PEG-Halos will aim to both improve upon the photophysical properties of the targeted voltage dyes as well as exploring the biological scope of HaloTag® system. Specically, this will include: 1) Further investigating the relationship between linker design and voltage sensitivity 2) Employing far-red voltage dyes to improve SNR and photostability 3) Genetic targeting of neuronal sub-types (excitatory, inhibitory) and sub-cellular domains (pre synaptic, post-synaptic) 4) Utilization of the RhoVR-PEG-Halos in more intact preparations (i.e. brain slice) 5) Development of a HaloTag®/GCaMP6s construct to enable simultaneous Ca2+ and voltage imaging
dc.relation.urlhttps://epostersonline.com/wep2019/node/62
dc.titleSynthesis of Red-Shifted Flourescent Voltage Sensors
dc.typePoster
dc.conference.dateJANUARY 13 - 17 , 2019
dc.conference.nameWEP Library ePoster competition 2019
dc.conference.locationKAUST
dc.contributor.institutionUC Berkeley
dc.contributor.institutionDalhousie University
refterms.dateFOA2019-06-26T11:38:14Z


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